EP1078076A2 - Farnesylgesteuerte endopeptidase - Google Patents

Farnesylgesteuerte endopeptidase

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Publication number
EP1078076A2
EP1078076A2 EP99917629A EP99917629A EP1078076A2 EP 1078076 A2 EP1078076 A2 EP 1078076A2 EP 99917629 A EP99917629 A EP 99917629A EP 99917629 A EP99917629 A EP 99917629A EP 1078076 A2 EP1078076 A2 EP 1078076A2
Authority
EP
European Patent Office
Prior art keywords
rcel
nucleic acid
polypeptide
substrate
mammalian
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99917629A
Other languages
English (en)
French (fr)
Inventor
Yun-Jung Choi
Anne K. North
George A. Martin
Gideon Bollag
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Onyx Pharmaceuticals Inc
Original Assignee
Onyx Pharmaceuticals Inc
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Filing date
Publication date
Application filed by Onyx Pharmaceuticals Inc filed Critical Onyx Pharmaceuticals Inc
Publication of EP1078076A2 publication Critical patent/EP1078076A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present mvention relates to all aspects of a farnesyl-directed endopeptidase. especially a mammalian farnes ⁇ 1-d ⁇ rected endopeptidase. such as human or mouse RCEl
  • An aspect of the invention is an isolated mammalian RCEl polypeptide or fragments of it. an isolated nucleic acid coding for a mammalian RCEl or fragments of it. and de ⁇ vatives of these polvpeptides and nucleic acids
  • Related polvpeptides. e g polvpeptides which are coded for by nucleic acids obtainable hybridization to a mammalian RCEl nucleic acid, are another feature of the invention
  • the invention also relates to methods of using such polvpeptides. nucleic acids, or de ⁇ vatives thereof, e g . m therapeutics, diagnostics, and as research tools, e g . to identify compounds which modulate a mammalian RCEl
  • the invention also concerns ligands of RCEl. - such as antibodies, nucleic acid aptamers, and substrates
  • Fig 1 shows a nucleotide and amino acid sequence of a human RCEl
  • Fig 2 shows a complete nucleotide sequence of mouse RCEl
  • Fig 3 shows a complete ammo acid sequence of mouse RCEl
  • Fig 4 shows a shows a compa ⁇ son between ammo acid sequences of human, mouse, and yeast RCEl A consensus sequence is shown Regions of ammo acid sequence identity are highlighted
  • Fig 5 shows a compa ⁇ son between the ammo acid sequences of human and mouse RCEl Regions of non-sequence identity are highlighted
  • an RCEl polypeptide has an amino acid sequence which is naturallv-obtamable and hich possesses at least one activity of the following an endoprotease activity, a substrate binding activity, a transformation- promoting activity, or an RCEl specific lmmunogenic activity
  • An endoprotease activity of RCEl means, for example, that the RCEl is capable of proteoh zing, or enzymatically cleavmg. a substrate at an internal ammo acid recognition site
  • the endoprotease activity is for a CAAX motif, where A is am aliphatic amino acid and
  • X is any amino acid
  • complete cleavage of the substrate results in the production of two fragments, each fragment having a termini defined by the ammo acid residue at the cleavage site, e g . -C-COOH and -NH 2 -A
  • the endoprotease activity is dependent upon the attachment of a hpid to the substrate (e g . at the cysteine residue), such as a cholesterol intermediate, e g.. a 15-carbon farnesyl or 20-carbon geranylgerany 1 moiety
  • Substrate binding is generalh considered the first step in enzyme catah sis because the substrate, acting as a ligand. must first attach to the enzyme surface to enable the enzyme to earn' out its catalytic reactions
  • This enzyme surface can be referred to as the active site of the enzvme Binding of the substrate to the enzyme surface can involve multiple interactions with the enzyme. e g .
  • a substrate binding activity as used herein means that a substrate attaches to the enzyme Attachment to the enzyme can be accomplished by one or more of the interactions which hold its naturally-occurnng substrate to it: however, a polypeptide can have a substrate binding activitv when it holds the substrate with less than the naturally-occurnng number and quality of interactions
  • Substrate bindmg and catalytic activity can be dissociated from each other
  • an RCEl polypeptide in accordance with the mvention can possess substrate binding activity but not an endoprotease activity
  • Substrate binding can optionally be effective to achieve catalysis of the substrate, to competitively or noncompetitively bind to the active site, to irreversibly attach to the enzyme, to result m the loss of catalytic activity (e g . where it is a suicide substrate), etc
  • the substrate comp ⁇ ses the CAAX motif
  • transformation-promoting activity/' it is meant an activity that produces a transformed phenotype of cells, e g . induces cell division, induces anchorage mdependent growth, increases ras activity, etc
  • the effect can be partial or incomplete
  • expression of a RCEl gene m cells can cause a transformed phenotype, or it can enhance the phenotype of already transformed cells lmmunogenic activity means that the polypeptide is capable of eliciting an immune response specific for an RCEl
  • the immune response can be a humoral (e g . induction of antibodies), cellular, or a combination thereof
  • the above-mentioned activities of an RCEl can be assayed, e g .
  • endoprotease activity can be measured as desc ⁇ bed in the examples below See also, e g . Methods in Enzymology. 250 251-266. 1995. Boyartchuk et al . Science. 275 1796. 1997
  • Substrate bindmg activity can be measured conventionally For mstance.
  • a competition binding assav can be emplo ed to identify substrates which attach to a polypeptide. or de ⁇ vative thereof, e g , b ⁇ combining under effective conditions, a substrate containing a detectable marker, an RCE 1 polypeptide.
  • the assay can be accomplished m liquid phase, where bound and free substrate is separated by a membrane, or. it can be accomplished in solid phase, as desired Solid-phase assays can be performed using high through-put procedures, e g , on chips, wafers, etc
  • a mammalian RCEl polypeptide is a mammalian polypeptide having an amino acid sequence which is obtainable from a natural source It therefore includes naturalK -occurring, normal, mutant, polymorphic, etc . ammo acid sequences which can be obtained from natural populations Natural sources include, e g .
  • the present invention also relates to fragments of a full-length mammalian RCEl polypeptide
  • the fragments are preferably biologically-active
  • biologically-active it is meant that the polypeptide fragment possesses an activity in a living s stem or with components of a living system
  • Biological- activities include those mentioned, e g . an endoprotease activity, a substrate binding activity, a transformation-promoting activity, and/or an lmmunogenic activity
  • Fragments can be prepared according to any desired method, including, chemical synthesis, genetic enginee ⁇ ng. cleavage products, etc See. below
  • the present invention also relates to a human RCEl having an amino acid sequence of amino acids 1 to 329, a va ⁇ ant containing contiguously ammo acids 1-230 and 252-329. ammo acids 231-251, amino acids 19-329 See, Fig 1
  • the 329-am ⁇ no acid polypeptide has a predicted molecular weight of about 35 8 kDa
  • RCEl sequences from another mammalian species, mouse has been cloned and identified These sequences include AA021859, AA072190. AA154658, AA154864, AA168614, AA218396. AA619282. AA790517, C77052. C86966,
  • the invention relates to a full-length mouse RCEl sequence as shown in
  • homologs from mammalian and non-mammalian can be obtained according to va ⁇ ous methods
  • hyb ⁇ dization with an ohgonucleotide (see below) selective for RCEl can be employed to select such homologs, e g . as desc ⁇ bed in Sambrook et al . Molecular Cloning, 1989. Chapter 11
  • Such homologs can have vary ing amounts of nucleotide and ammo acid sequence identity and simila ⁇ ty to RCE 1
  • Non-mammalian organisms include, e g . vertebrates, invertebrates, zebra fish, chicken. Drosophila. C elegans. roundworms. prokarvotes. plants. Arabidopsis. viruses. etc
  • the invention also relates to RCEl specific or unique amino acid sequences, e g , a defined amino acid sequence which is found in the particular RCE 1 sequence but not m another amino acid sequence
  • RCEl specific or unique amino acid sequences e g , a defined amino acid sequence which is found in the particular RCE 1 sequence but not m another amino acid sequence
  • a specific ammo acid sequence can be found routinely, e g . by searchmg a gene/protein database using the BLAST set of computer programs
  • Such specific sequences include, e g . human and mouse but not yeast RCEl. human but not mouse or yeast RCEl. mouse but not human or yeast
  • RCEl Human and mouse RCEl specific sequences include e g , AALGGD, TGIQPGT, MQLSMDCPCD. DGLKW. ARCLTDMRWL. LVFRACM. RFRQSSVG. and PKLYGS See
  • a mouse or human RCE 1 specific or umque amino acid sequence when possessing an lmmunogenic activity, can be useful to produce peptides as antigens to generate an immune response specific for RCE Antibodies obtained by such immunization can be used as a specific probe for RCE protein for diagnostic or research purposes
  • a polypeptide of the invention e g . having a polypeptide sequence as shown in Fig 1 and Fig 3 can by analyzed by available methods to identify structural and/or functional domains in the polypeptide
  • the polypeptide coding sequence set forth in Fig 1 is analyzed by hydropathy and hydrophihcity analysis (e g , Kyte and Doohttle, J Mol Bio ,157 105, 1982) putative membrane spanning regions are identified at A25-W56. F72-W89. L109-M136. A181- F209, V223-I249. T251-L276, and L284-L302 Vanous other programs can be used to analyze its structure and routmely predict functional domains, including. EMBL Protein Predict. Rost and Sander, Protems, 19 55-72, 1994
  • a polypeptide of the present invention can also have 100% or less ammo acid sequence identity to the ammo acid sequence set forth m Fig 1
  • Sequence identity means that the same nucleotide or amino acid which is found in the sequence set forth in Fig 1. Fig 2. or Fig 4 is found at the corresponding position of the compared sequence(s).
  • a polypeptide having less than 100% sequence identify' to the ammo acid sequence set forth in Fig 1 or 3 can contain various substitutions from the naturally-occurnng sequence, including homologous amino acid substitutions See below for examples of homologous ammo acid substitution
  • the sum of the identical and homologous residues divided by the total number of residues in the sequence over which the RCEl polypeptide is compared is equal to the percent sequence simila ⁇ ty
  • the compared sequences can be aligned and calculated according to any desired method, algo ⁇ thm. computer program, etc , including, e g . FASTA.
  • BLASTA A polypeptide having less than 100% amino acid sequence identity to the ammo acid sequence of Fig 1 can compnse e g . about 99%. 97%. 95%. but greater than 35% identity A prefe ⁇ ed amount of sequence identity is about greater than 94% (e g , human and mouse exhibit 94% sequence identity)
  • a RCEl polypeptide. fragment, or substituted polypeptide can also compnse vanous modifications, here such modifications include pid modification such as prenylation (e g . 15- carbon farnesyl. 20-carbon geranylgeranyl) or other cholesterol intermediates and denvatives. methylation. phosphorylation. glycosylation. covalent modifications (e g . of an R-group of an ammo acid), ammo acid substitution, amino acid deletion, or amino acid addition Modifications to the polypeptide can be accomplished according to various methods, including recombinant. synthetic, chemical, etc
  • a mutation to a RCEl polypeptide can be selected to have a biological activity of RCEl. e g . an endoprotease activity, a substrate bmding activity, a transformation-promoting activity, or an lmmunogenic activity
  • a biological activity of RCEl. e g . an endoprotease activity, a substrate bmding activity, a transformation-promoting activity, or an lmmunogenic activity The selection and preparation of such mutations is discussed below
  • Polvpeptides of the present invention can be used in vanous ways, e g , m assays, as lmmunogens for antibodies as desc ⁇ bed below, as biologically-active agents (e g . having one or more of the activities associated with RCEl) Fragments having ras substrate bindmg activity, and optionally lacking other biological activities, can be utilized to block ras processmg Such fragments can be administered as DNA (e g , m vectors, naked DNA. etc ) or they can be administered in forms that can penetrate cells, e g . m liposomes.
  • DNA e g , m vectors, naked DNA. etc
  • a useful fragment can be identified routinely by testing the ability of overlapping fragments of the entire length of RCEl to inhibit an RCEl activity The measurement of these activities is desc ⁇ bed below and in the examples These peptides can also be identified and prepared as descnbed in EP 496 162 Peptides can be chemically-modified, etc
  • An RCEl polypeptide, a denvative thereof, or a fragment thereof, can be combmed with one or more structural domams, functional domams. detectable domains, antigenic domains, and/or a desired polvpeptides of interest, in an arrangement which does not occur m nature, l e . not naturally- occur ⁇ ng. e g .
  • a genomic fragment prepared from the genome of a living organism, e g , an animal, preferably a mammal, such as human, mouse, or cell lines thereof
  • a polypeptide compnsmg such features is a chimenc or fusion polypeptide
  • Such a chime ⁇ c polypeptide can be prepared according to vanous methods, including, chemical, synthetic, quasi- synthetic. and/or recombinant methods
  • a chime ⁇ c nucleic acid coding for a chime ⁇ c polypeptide can contain the vanous domains or desired polvpeptides in a continuous or interrupted open reading frame, e g . containing mtrons.
  • a domain or desired polypeptide can possess any desired propert . including, a biological function such as catalytic. signalling, growth promoting, cellular targeting (e g , signal sequence, targeting sequence, such as to endosomes, lysosomes. ER. nucleus), etc . a structural function such as hydrophobic, hydrophilic. membrane-spanmng. etc . receptor-ligand functions, and/or detectable functions, e g . combined with enzyme, fluorescent polypeptide. green fluorescent protein. (Chalfie et al . 1994. Science.
  • an RCEl polypeptide. or a part of it can be used as selectable marker when introduced into a host cell
  • a nucleic acid codmg for an ammo acid sequence according to the present invention can be fused in frame to a desired codmg sequence and act as a tag for pu ⁇ fication. selection, or marking purposes
  • the region of fusion can encode a cleavage site to facilitate expression, isolation, punfication.
  • a polypeptide accordmg to the present mvention can be produced in an expression system, e g .
  • Modifications to the polypeptide imparted by such system include, glycosylation, ammo acid substitution (e g , by diffenng codon usage), polypeptide processing such as digestion, cleavage, endopeptidase or exopeptidase activity, attachment of chemical moieties, including pids (prenylation), phosphates, etc
  • a polypeptide according to the present invention can be recovered from natural sources, transformed host cells (culture medium or cells) according to the usual methods, including, detergent extraction (e g , CHAPSO, octylglucoside). ammonium sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography. phosphocellulose chromatography, hydrophobic interaction chromatography, hy droxyapatite chromatography and lectin chromatography Protein refolding steps can be used, as necessary, in completing the configuration of the mature protein Finally, high performance liquid chromatography (HPLC) can be employed for final pu ⁇ fication steps
  • a mammalian RCEl nucleic acid, or fragment thereof is a nucleic acid having a nucleotide sequence obtamable from a natural source, or compnsing a naturallv-obtamable coding sequence for a mammalian RCEl polypeptide See. above It therefore includes naturally-occurnng sequences from normal, mutant, polymorphic, degenerate sequences, etc . alleles which can be obtained from natural populations Natural sources include, e g . living cells obtained from tissues and whole organisms, cultured cell lines, including p ⁇ mary and immortalized cell lines Human RCEl is expressed in. e g , heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancrease.
  • spleen thvrnus. prostate, testis. ovary, small intestine, colon, and pe ⁇ pheral blood leucocytes It is also expressed in vanous cancer cells, including. HL-60. Hela cell S3, chronic mvelogenous leukemia K- 562. lymphoblastic leukemia MOLT-4. Burkitt s lymphoma Raji. colorectal adenocarcinoma SW 480. lung carcinoma A549, and melanoma G361
  • a nucleic acid sequence of a human allele of RCEl is shown in Fig 1 contains an open- reading frame of 329 ammo acids at nucleotide positions 32 to 1021
  • a splice vanant of such nucleic acid is also illustrated in Fig 1, containing an open-reading frame of 308 amino acids at nucleotide positions to 32 to 722 and 786-1021
  • the invention also relates to nucleotides 723 to 785 (useful fragments thereof), absent in the splice vanant. which can be used, e g .
  • a nucleic acid sequence of the invention can contain the complete coding sequence from ammo acid 1 to amino acid 329, degenerate sequences thereof, and fragments thereof
  • a nucleic acid according to the present invention can also compnse a nucleotide sequence which is 100% complementary, e g . an anti-sense, to any nucleotide sequence mentioned above and below
  • a nucleic acid accordmg to the present invention can be obtained from a va ⁇ ety of different sources It can be obtained from DNA or RNA. such as polyadenylated mRNA. e g , isolated from tissues, cells, or whole organism The nucleic acid can be obtained directly from DNA or RNA.
  • the nucleic acid can be obtamed from a cell at a particular stage of development, having a desired genotype, phenotype (e g . an oncogemcally transformed cell or a cancerous cell), etc
  • a nucleic acid comp ⁇ sing a nucleotide sequence coding for a polypeptide accordmg to the present invention can mclude only a coding sequence of an RCEl. a coding sequence of an RCEl and additional codmg sequence (e g , sequences codmg for leader, secretory, targeting, enzvmatic. fluorescent or other diagnostic peptides). coding sequence of RCEl and non-coding sequences, e g . untranslated sequences at either a 5' or 3' end.
  • nucleic acid compnsing a nucleotide sequence codmg without mtemiption for an RCEl polypeptide means that the nucleotide sequence contains an ammo acid codmg sequence for an RCEl polypeptide, with no non-coding nucleotides interrupting or intervening in the coding sequence, e g . absent ⁇ ntron(s)
  • Such a nucleotide sequence can also be desc ⁇ bed as contiguous
  • a genomic DNA codmg for an RCEl can be obtained routinelv
  • a nucleic acid according to the present invention also can comp ⁇ se an expression control sequence operably linked to a nucleic acid as descnbed above
  • expression control sequence means a nucleic acid sequence which regulates expression of a polypeptide coded for bv a nucleic acid to which it is operably linked Expression can be regulated at the level of the mRNA or polypeptide
  • the expression control sequence includes mRNA-related elements and protein- related elements Such elements mclude promoters, enhancers (viral or cellular), ⁇ bosome binding sequences, transc ⁇ ptional terminators, etc
  • An expression control sequence is operablv linked to a nucleotide coding sequence when the expression control sequence is positioned in such a manner to effect or achieve expression of the codmg sequence
  • expression of the coding sequence is d ⁇ ven by the promoter
  • Expression control sequences can be heterologous or endogenous to the normal gene
  • a nucleic acid in accordance with the present invention can be selected on the basis of nucleic acid hyb ⁇ dization
  • the ability of two single-stranded nucleic acid preparations to hybndize together is a measure of their nucleotide sequence complementanty. e g , base-pai ⁇ ng between nucleotides, such as A-T.
  • the mvention thus also relates to nucleic acids which hvb ⁇ dize to a nucleic acid comp ⁇ smg a nucleotide sequence as set forth m Fig 1 and Fig 2
  • a nucleotide sequence hvbndizmg to the latter sequence ill have a complementarv nucleic acid strand, or act as a template for one in the presence of a polymerase (I e . an appropnate nucleic acid synthesizing enzvme)
  • the present mvention mcludes both strands of nucleic acid, e g . a sense strand and an anti-sense strand
  • Hybridization conditions can be chosen to select nucleic acids which have a desired amount of nucleotide complementanty with the nucleotide sequence set forth in Fig 1 or 2
  • a nucleic acid capable of hybndizmg to such sequence preferably, possesses 85%, 90%, more preferably 95%, 99%. or more, complementanty, between the sequences
  • the present invention particularly relates to
  • stnngent conditions means any conditions in which h bndization will occur where there is at least about 85%. about 94%. preferably 97%, nucleotide complementanty between the nucleic acids Stnngent conditions include 50% formamide. 6X SSC or 6X SSPE. and optionally, a blockmg agent (s)s (e g .
  • Hybndization can be performed as desc ⁇ bed m Sambrook et al . Molecular Cloning. 1989. Chapter 9 Hybndization can also be based on calculation of the melting temperature (Tm) of the hyb ⁇ d formed bet een the probe and its target, as descnbed m Sambrook et al Nucleic acids which are preferably excluded are AA021859, AA072190, AA154658, AA154864, AA168614,
  • yeast RCEl or a fragment of yeast RCEl
  • a nucleic acid or polypeptide can comp ⁇ se one or more differences in the nucleotide or amino acid sequence set forth in Fig 1 , 2. or 3 Changes or modifications to the nucleotide and or amino acid sequence can be accomplished by any method available, including directed or random mutagenesis
  • a nucleic acid codmg for an RCEl according to the invention can comp ⁇ se nucleotides which occur in a naturally-occurnng RCE 1 gene e g . naturally-occurnng polymorphisms, normal or mutant alleles (nucleotide or ammo acid), mutations which are discovered in a natural population of mammals, such as humans, monkeys, pigs. mice. rats, or rabbits
  • Naturally- occurnng it is meant that the nucleic acid is obtainable from a natural source, e g . animal tissue and cells, body fluids, tissue culture cells, forensic samples
  • Naturally-occurnng mutations to RCEl can include deletions (e g .
  • a nucleotide sequence codmg for a RCEl polypeptide of the invention can contain codons found in a naturally-occurnng gene, transc ⁇ pt. or cDNA. for example, e g . as set forth in Fig 1, 2, or 3, or it can contain degenerate codons codmg for the same ammo acid sequences
  • Modifications to an RCE 1 sequence, e g , mutations can also be prepared based on homology searching from gene data banks, e g , Genbank.
  • EMBL Sequence homology searching can be accomplished usmg vanous methods, including algonthms descnbed m the BLAST family of computer programs, the Smith-Waterman algonthm. etc
  • homologous amino acids can be identified between vanous sequences, such as the human and yeast RCEl and used as the basis to make ammo acid substitutions See. e g , Fig 2
  • a mutat ⁇ on(s) can then be introduced into an RCEl sequence by identifying and aligning ammo acids conserved between the polvpeptides and then modifying an ammo acid m a conserved or non-conserved position
  • a nucleic acid and corresponding polypeptide of the present mvention include sequences which differ from the nucleotide sequence of Fig 1 or Fig 2 but which are phenotypically silent
  • sequence modifications mclude. e g , nucleotide substitution which do not affect the ammo acid sequence (e g . different codons for the same amino acid or degenerate sequences), replacmg naturally-occurnng amino acids with homologous ammo acids, e g , (based on the size of the side cham and degree of polanzation) small nonpolar cvsteine, pro ne. alanine. threonine. small polar senne, glycme, aspartate. asparagine.
  • Homologous acids can also be grouped as follows uncharged polar R groups, glvcme. senne. threonme. cysteme. ty rosine. asparagme. glutamine. acidic amino acids (negatively charged), aspartic acid and glutamic acid, basic ammo acids (positively charged), lvsine. argmme. histidine Homologous ammo acids also include those desc ⁇ bed bv Dav hoff in the Atlas of Protem Sequence and Structure 5 ( 1978). and by Argos in EMBO J . 8. 779-785 (1989)
  • a nucleic acid can comp ⁇ se a nucleotide sequence coding for a polypeptide having an ammo acid sequence as set forth in Fig 1 or Fig 3. except where one or more positions are substituted by conservative ammo acids, or a nucleotide sequence coding for a polypeptide havmg an amino acid sequence as set forth m Fig 1 or 3. except having 1, 5. 10. 15. or 20 substitutions, e g . wherein the substitutions are conservative amino acids
  • the invention also relates to polvpeptides coded for by such nucleic acids In addition, it may be desired to change the codons in the sequence to optimize the sequence for expression in a desired host
  • a nucleic acid according to the present invention can comp ⁇ se. e g . DNA. RNA. synthetic nucleic acid, peptide nucleic acid, modified nucleotides. or mixtures
  • a DNA can be double- or single-stranded
  • Nucleotides comp ⁇ smg a nucleic acid can be joined via vanous known linkages, e g , ester, sulfamate, sulfamide. phosphorothioate, phosphoramidate, methylphosphonate, carbamate, etc , depending on the desired purpose, e g . resistance to nucleases. such as RNase H. improved in vivo stability, etc See. e g . U S Pat Nos 5.378,825
  • Vanous modifications can be made to the nucleic acids, such as attaching detectable markers (avidin. biotin. radioactive elements), moieties which improve hybndization. detection, or stability
  • the nucleic acids can also be attached to solid supports, e g , nitrocellulose, magnetic or paramagnetic microspheres (e g , as desc ⁇ bed m U S Pat No 5.411.863. U S Pat No 5.543.289. e g , comp ⁇ smg fenomagnetic, supermagnetic. paramagnetic, superparamagnetic. iron oxide and polysaccha ⁇ de). nylon, agarose. diazotized cellulose, latex solid microspheres. polyacrylamides. etc . according to a desired method See, e g , U S Pat Nos 5,470.967. 5.476,925. 5,478.893
  • ohgonucleotides and nucleic acid probes Such ohgonucleotides or nucleic acid probes can be used, e g . to detect, quantitate. or isolate a RCEl nucleic acid in a test sample Detection can be desirable for a va ⁇ ety of different purposes, mcluding research, diagnostic, and forensic For diagnostic purposes, it may be desirable to identify the presence or quantity of a RCEl nucleic acid sequence m a sample, where the sample is obtamed from tissue, cells, body fluids, etc. In a preferred method, the present invention relates to a method of detecting a RCEl nucleic acid comp ⁇ s g.
  • ohgonucleotide in accordance ith the invention can also be used in synthetic nucleic acid amplification such as PCR (e g . Saiki et al . 1988. Science. 241 53. U S Pat No 4.683.202. PCR Protocols A Guide to Methods and Applications. Innis et al . eds . Academic Press, New York. 1990) or differential display (See. e g Liang et al . Nucl Acid Res . 21 3269-3275. 1993. U S Pat No 5.599.672. W097/18454)
  • Useful ohgonucleotides include, e g .nucleotides 723-785 of Fig 1.
  • nucleotide sequence which is unique to RCEl
  • a unique sequence to RCEl it is meant a defined order of nucleotides which occurs in RCEl. e g . m the nucleotide sequence of Fig 1 or Fig 2, but rarely or infrequently m other nucleic acids. especially not in an animal nucleic acid, preferably mammal, such as human, rat.
  • a umque nucleic acid according to the present invention can be determined routinely A nucleic acid compnsing a unique sequence of RCEl can be used as a hyb ⁇ dization probe to identify the presence of RCEl m a sample comp ⁇ sing a mixture of nucleic acids, e g . on a Northern blot H b ⁇ dization can be performed under stnngent conditions to select nucleic acids having at least 95% identity (I e .
  • RCEl nucleotide sequence can also be fused in-frame, at either its 5' or 3' end. to vanous nucleotide sequences as mentioned throughout the patent, including coding sequences for other parts of RCEl. enzvmes. GFP. etc. expression control sequences, etc Hybndization can be performed under different conditions, dependmg on the desired selectivity, e g . as descnbed in Sambrook et al . Molecidar Cloning, 1989 For example, to specifically detect RCEl.
  • an ohgonucleotide can be hybndized to a target nucleic acid under conditions in which the ohgonucleotide only hy b ⁇ dizes to RCEl, e g . where the ohgonucleotide is 100% complementary to the target
  • Different conditions can be used if it is desired to select target nucleic acids which have less than 100% nucleotide complementanty. at least about, e g . 99%. 97%,
  • an ohgonucleotide according to the present invention can be used diagnostically
  • a patient having symptoms of a cancer or other condition associated with the Ras signaling pathway can be diagnosed with the disease by using an ohgonucleotide according to the present invention, in polymerase chain reaction followed bv DNA sequencing to identify whether the sequence is normal, in combmation with other ohgonucleotides to oncogenes or genes in the ras signalling pathway, etc .
  • the present invention relates to a method of diagnosing a cancer compnsing contacting a sample compnsing a target nucleic acid with an ohgonucleotide under conditions effective to permit hybndization between the target and ohgonucleotide. detecting hybndization. wherein the ohgonucleotide compnses a sequence of RCEl. preferably a unique sequence of.
  • the sequence can be determined according to vanous methods, including isolating the target nucleic acid, or a cDNA thereof, and determining its sequence according to a desired method
  • Ohgonucleotides (nucleic acid) accordmg to the present invention can be of any desired size. e g , about 10-200 nucleotides. 12-100. preferably 12-50. 12-25. 14-16, at least about 15. at least about 20. etc
  • Such ohgonucleotides can have non-naturally-occurnng nucleotides. e g . inosine
  • Such ohgonucleotides have 100% identity or complementanty to a sequence of Fig 1 or Fig 2, or it can have mismatches or nucleotide substitutions, e g , 1, 2, 3, 4, or 5 substitutions
  • the ohgonucleotide can comp ⁇ se a kit.
  • kits include a desired buffer (e g , phosphate, tns, etc ), detection compositions, etc
  • a desired buffer e g , phosphate, tns, etc
  • the ohgonucleotide can be labeled or unlabeled. with radioactive or non-radioactive labels as known m the art
  • Anti-sense nucleic acid can also be prepared from a nucleic acid according to the present, preferably an anti-sense to a coding sequence of Fig 1, 2, or 3
  • Antisense nucleic acid can be used in vanous ways, such as to regulate or modulate expression of RCEl. e g . inhibit it.
  • an anti-sense ohgonucleotide can be operably linked to an expression control sequence
  • the nucleic acid according to the present mvention can be labelled accordmg to any desired method
  • the nucleic acid can be labeled usmg radioactive tracers such as 32 P. 35 S. I25 1. 3 H. or 14 C. to mention only the most commonly used tracers
  • the radioactive labelling can be earned out accordmg to any method such as. for example, terminal labeling at the 3' or 5' end usmg a radiolabeled nucleotide.
  • a non-radioactive labeling can also be used, combining a nucleic acid of the present invention with residues having lmmunological properties (antigens, haptens), a specific affinity for certain reagents (ligands), properties enabling detectable enzyme reactions to be completed (enzymes or coenzymes. enzyme substrates, or other substances involved in an enzymatic reaction), or characteristic physical properties, such as fluorescence or the emission or absorption of light at a desired wavelength, etc
  • a nucleic acid accordmg to the present invention including ohgonucleotides. anti-sense nucleic acid, etc . can be used to detect expression of RCEl in whole organs, tissues, cells, etc . bv vanous techniques, including Northern blot. PCR. in situ hybndization. etc Such nucleic acids can be particularly useful to detect disturbed expression, e g , cell-specific and/or subcellular alterations, of RCEl The levels of RCEl can be determined alone or in combination ith other genes products (oncogenes such as Ras). transc ⁇ pts. etc
  • a nucleic acid according to the present invention can be expressed m a va ⁇ ety of different systems, in vitro and in vivo, according to the desired purpose
  • a nucleic acid can be inserted into an expression vector, introduced into a desired host, and cultured under conditions effective to achieve expression of a polypeptide coded for the nucleic acid
  • Effective conditions includes any culture conditions which are suitable for achieving production of the polypeptide by the host cell, including effective temperatures. pH, medias. additives to the media in which the host cell is cultured (e g . additives which amplify or induce expression such as but> ⁇ ate. or methotrexate if the coding nucleic acid is adjacent to a dhfr gene), cyclohexamide.
  • a nucleic acid can be introduced mto the cell by any effective method including, e g . naked DNA. calcium phosphate precipitation, electroporation. injection. DEAE-Dextran mediated transfection. fusion with liposomes. associated with agents which enhance its uptake into cells, viral transfection
  • a cell mto which a nucleic acid of the present invention has been introduced is a transformed host cell
  • the nucleic acid can be extrachromosomal or integrated into a chromosome(s) of the host cell It can be stable or transient
  • An expression vector is selected for its compatibility with the host cell
  • Host cells include, mammalian cells, e g . COS-7. CHO. HeLa. LTK.
  • yeast insect cells, such as Sf9 (S frugipeda). High Five Cells (Invitrogen). Drosophila. bacte ⁇ a. such as E coh. Streptococcus, bacillus, yeast, fungal cells, plants, embryonic stem cells (e g . mammalian, such as mouse or human), cancer or tumor cells Sf9 are preferred for insect expression, expression can be accomplished according to. e g . O'Reilly et al . Baculovirus Expression Vectors A Laboratory Manual, Freeman, NY, 1992 HEK293 mammalian cells can be used for mammalian overexpression See. e g. Collins et al .
  • Expression control sequences are similarly selected for host compatibility and a desired purpose, e g , high copy number, high amounts, induction, amplification, controlled expression
  • Other sequences which can be employed include enhancers such as from SV40. CMV. RSV. inducible promoters, cell-type specific elements, or sequences which allow selective or specific cell expression
  • Promoters that can be used to d ⁇ ve expression include, e g . the endogenous promoter. MMTV, SV40, tip. lac. tac. or T7 promoters for bacte ⁇ al hosts, or alpha factor, alcohol oxidase. or PGH promoters for yeast
  • Another gene of interest can be introduced mto the same host for purposes of. e g . modulating expression RCEl. elucidating RCEl function or that of the gene of interest Genes of interest include other oncogenes. genes involved in the cell cycle, etc Such genes can be the normal gene, or a va ⁇ ation, e g , a mutation, chimera, polymorphism, etc
  • a nucleic acid or polypeptide of the present invention can be used as a size marker in nucleic acid or protein electrophoresis. chromatography'. etc Defined rest ⁇ ction fragments can be determined by scanning the sequence for restnction sites, calculating the size, and performing the conesponding restnction digest
  • the RCEl polypeptide can also be used as a 35 8 kd molecular weight marker for a protein gel
  • the RCEl DNA disclosed herein can also be used as a 1472 bp marker on a DNA gel
  • Another aspect of the present invention relates to the regulation of biological pathways in which a RCEl gene is involved, particularly pathological conditions
  • cell proliferation e g . cancer
  • growth control e g . growth control
  • mo ⁇ hogenesis. . 268 233- 239. 1995 Bussev. Science, 272 225-226,
  • RCEl is involved in the ras-dependent signal-transduction cascade It is responsible for COOH-terminal processing of ras.
  • a step in ras maturation Over-expression of ras leads to oncogenic activity
  • One approach to treatmg ras over-expression is inhibiting the ras maturation pathway so incompletely processed and inactive ras accumulates, eliminating or reducing its oncogemc effect
  • the ras maturation pathway can be inhibited by blocking RCEl activity Such blocking can be accomplished in vanous ways, including by admmiste ⁇ ng RCEl antibodies or other ligands.
  • RCEl peptides especially those that bind to the CAAX motif but lack endoproteolytic activity
  • inhibitors of RCEl endoprotease anti-sense or double-stranded RNA (e g . Fire et al , Nature. 391 806-811. 1998)
  • Blocking agents can be identified according to the methods desc ⁇ bed herein or those available in the art
  • RCEl activity can be modulated by mcreasmg. reducing, antagonizing, promoting, stabilizing, etc RCEl In one method of the invention.
  • RCEl activity can be measured by reacting, m the presence of a test compound, a substrate comp ⁇ smg a CAAX polypeptide motif and a mammalian RCEl.
  • a substrate that can be enzymaticallv digested. 1 e . proteolytically removed, by RCE 1 preferably compnses a CAAX recognition site, where an RCEl cleaves between the cysteme and aliphatic amino acid residues, prenylated CAAX containing peptides. such as a farnsylated. or geranylgeranylated CAAX peptides.
  • Any substrate is suitable if it can be acted upon by RCEl
  • a substrate can compnse other atoms, such as additional amino acid residues linked by peptide or other bonds, and can be modified in any desirable way
  • a substrate can be affixed to a solid support.
  • a substrate can also be detectably labeled, e g . with antibody, avidm. biotin. radioactive labels, aptamers. fluorescent labels, nucleic acid, etc
  • the substrate can also comp ⁇ se phosphates, methy 1 groups, sugars, or hpids In a preferred embodiment, the substrate contains a hpid. e g .
  • the substrate is prenylated
  • the substrate is b ⁇ ot ⁇ n-Lys-Lys-Ser-Lys-Thr-Lys-(Farnesyl)Cys-Val-Ile-Met.
  • test compound is preferably reacted with an RCEl in a milieu in which RCEl cleaves the substrate
  • a milieu can be referred to as effective conditions
  • effective reaction conditions can be determined in the absence of the test compound to establish a baseline activity, e g., as in a control
  • the effective reaction conditions can be routmely selected, e g , usmg salts, buffers, reducmg and/or oxidizing agents. pH's. etc W en utilizing a substrate comp ⁇ smg a CAAX motif, effective cleavage results in the removal of the AAX residues from the substrate, exposing the Cys-COOH terminus
  • proteolysis detection mvolves identifying a product of the reaction For example, when the cleavage site is an ammo acid sequence, complete proteolysis of the substrate results in cleavage products having novel 3' and 5' termini The products can be detected directly, e g , by chromatography, electrophoresis, mass spectroscopy, lmmunoassay etc , or the termini can be detected, e g .
  • the substrate compnses CAAX and cleavage results in the appearance of the Cys-COOH termini the latter is detected by methvlatmg it using a methylase and a labeled-methiomne-substrate
  • the methylase is a prenyl protein-specific methyltransferase (PPSMT) and the methionine- substrate is ⁇ -S-adenosyl methionme.
  • PPSMT prenyl protein-specific methyltransferase
  • the methionine- substrate is ⁇ -S-adenosyl methionme.
  • the RCEl substrate is labeled at its 5' end with biotin. it can be captured by avidin which is preferably attached to beads.
  • the RCEl substrate can be attached to a solid surface, a magnetic bead. etc. and processed conventionally.
  • a methylase can be purified, enriched, provided as a component of a cell extract, e.g., from a mammalian cell or yeast cell. etc.
  • the extract or lysate can be obtained from various cells, including cells transformed with a methylase gene. e.g.. yeast STE14. See. e.g., Hrycyna et al.. Methods in Enzymology, 250:251-266, 1995.
  • the RCEl component i.e., a polypeptide or endoproteolytic fragment thereof
  • the RCEl component can be added to the reaction mixture in a variety of forms, e.g., substantially purified, as a component of cell membranes (such as. endoplasmic reticulum). or as a soluble extract.
  • the RCEl polypeptide can be obtained from a natural source, a recombinant source, or it can be produced synthetically (produced chemically or enzymatically. e.g.. cleavage of a full-length RCEl).
  • the RCEl is expressed in a cell line transformed with an RCEl coding sequence (e.g.. a cDNA. a gene, a genomic fragment, etc.).
  • the RCEl is present as a heterologous component of the cell: by heterologous. it is meant that the RCEl is not only expressed in a cell line of a different species, but it is also coded for by a coding sequence that has been introduced into the cell, e.g., by transfection, transformation, etc.
  • the RCEl is expressed at high levels in the cell.
  • a human RCEl. or a fragment thereof, is a preferred coding sequence. See. e.g., Fig. 1.
  • a useful fragment of RCEl comprises an endoprotease activity and substrate binding activity, e.g. amino acids 19-329.
  • the RCEl is provided as a cell lysate, e.g.. cells transformed with RCEl are lysed and the resulting lysate is used directly in the assay, i.e.. a crude lysate.
  • the crude lysate comprising the recombinant RCEl can optionally be refined or enriched for RCEl. For instance, e.g. a membrane fraction can be isolated, etc.
  • RCEl (such as HEK293) are harvested, washed in PBS + 20 mM EDTA. lysed by douncing in hypotonic lysis buffer or by using nitrogen cavitation. subjected to a low speed spin to remove insoluble material and cell debris (including unbroken cells and nuclei), and then centrifuged at lOO.OOOg for an amount of time effective to pellet membranes.
  • a pu ⁇ ose of the assay is to select and identify compounds which modulate RCEl activity.
  • proteolysis detection is typically performed in the presence and absence of the test compound. Whether a compound modulates RCEl activity can be determined routinely, e.g., by determining whether more or less proteolysis has occurred in the presence of the test compound.
  • the assay can also be conducted in yvhole cells For example, cells over-expressing an RCEl have a transformation promoting activity Over-expression can be achieved in a cell by genetic enginee ⁇ ng means, e g . transforming an RCEl gene operablv linked to a robust promoter, bv selecting cell lines (such as HEK293) for such activity, etc Agents can be administered to such cells and tested for their ability to inhibit transformation, e g . by momto ⁇ ng cell mo ⁇ hology. etc
  • Compounds identified in this or other manners can be useful to modulate RCEl activity in a cell, a tissue, a yvhole organism, in situ, in vitro (test tube, a solid support, etc ). in vivo, or in any desired environment
  • a compound having such an in vitro activity ill be useful in vivo to modulate a biological pathway associated with RCEl.
  • e g to treat a pathological condition associated with the biological and cellular activities mentioned above
  • the present invention thus also relates to the treatment and prevention of diseases and pathological conditions associated with ras-mediated signal transduction. e g .
  • the mvention relates to a method of treating cancer compnsmg administe ⁇ ng, to a subject in need of treatment, an amount of a compound effective to treat the disease, where the compound is a regulator of RCElgene or polypeptide expression Treating the disease can mean, delaying its onset, delaying the progression of the disease, improving or delaying clinical and pathological signs of disease
  • a regulator compound, or mixture of compounds can be synthetic, naturally-occurnng, or a combination
  • a regulator compound can comp ⁇ se ammo acids, nucleotides, hydrocarbons, hpids. polysaccha ⁇ des, etc
  • a regulator compound is preferably a regulator of RCEl. e g .
  • protein expression, or processing Expression can be regulated using different agents, e g , an anti-sense nucleic acid, a nbozvme. an aptamer. a synthetic compound, or a naturally-occurnng compound
  • agents e g , an anti-sense nucleic acid, a nbozvme. an aptamer. a synthetic compound, or a naturally-occurnng compound
  • the compound, or mixture can be formulated mto pharmaceutical composition compnsmg a pharmaceutically acceptable earner and other excipients as apparent to the skilled worker See, e g , Remington's Pharmaceutical Sciences. Eighteenth Edition. Mack Pubhshmg Company.
  • composition can additionally contam effective amounts of other compounds, especially for treatment of cancer
  • the present invention also relates to antibodies which specifically recognize a RCEl polypeptide Antibodies, e g , polv clonal. monoclonal, recombinant, chimenc. can be prepared according to any desired method
  • a polypeptide according to Fig 1. can be administered to mice, goats, or rabbit subcutaneouslv and/or mtrape ⁇ toneally. with or without adjuvant, in an amount effective to elicit an immune response
  • the antibodies can also be single chain or FAb
  • the antibodies can be IgG, subtypes. IgG2a. IgGl. etc
  • Antibodies can also be generated bv administenng naked DNA See. e g . U S Pat Nos 5.703.055. 5.589.466, 5.580,859
  • an antibody specific for RCE 1 means that the antibody recognizes a defined sequence of amino acids within or including the RCEl ammo acid sequence of Fig 1 or Fig 3 Thus, a specific antibody will bmd with higher affinity to an amino acid sequence. 1 e . an epitope. found in Fig 1 or 3 than to ep ⁇ tope(s) found in a different protein, e g . as detected and/or measured by an lmmunoblot assay
  • an antibody which is specific for an epitope of RCEl is useful to detect the presence of the epitope m a sample, e g . a sample of tissue containing RCEl gene product, distinguishing it from samples in which the epitope is absent
  • Such antibodies are useful as desc ⁇ bed in Santa Cruz
  • ligands which bind to an RCEl polypeptide accordmg to the present invention. or a de ⁇ vative thereof can also be prepared, e g . using synthetic peptide branes or aptamers (e g .
  • Antibodies and other ligands which bmd RCEl can be used in vanous ways, including as therapeutic, diagnostic, and commercial research tools, e g. to quantitate the levels of RCEl polypeptide in animals, tissues, cells, etc , to identify the cellular localization and/or distribution of RCEl. to pu ⁇ fy RCEl. or a polypeptide compnsing a part of RCEl, to modulate the function of RCEl. etc
  • Antibodies to RCEl. or a de ⁇ vative thereof can be used in Western blots. ELIZA, lmmunoprecipitation. RIA. etc
  • the present mvention relates to such assays, compositions and kits for performing them, etc Similarly, antibodies that bmd RCEl can be used to immunoprecipitate
  • An antibody according to the present mvention can be used to detect RCEl polypeptide or fragments thereof m vanous samples, including tissue, cells, body fluid, blood, u ⁇ ne. cerebrospmal fluid
  • a method of the present invention compnses contacting a ligand which binds to a peptide of Fig 1 or 3 under conditions effective, as known in the art. to achieve binding, detecting specific binding between the ligand and peptide
  • specific bmdmg it is meant that the ligand attaches to a defined sequence of ammo acids, e g . withm or including the ammo acid sequence of Fig 1 or Fig 3
  • the antibodies or denvatives thereof can also be used to inhibit expression of RCEl or a fragment thereof
  • the levels of RCEl polypeptide can be determined alone or in combination with
  • the amount (e.g.. its expression level) of RCEl polypeptide can be compared (e.g., as a ratio) to the amounts of other polvpeptides in the same or different sample, e.g.. ras. Ftase, etc.
  • a ligand for RCEl can be used in combmation with other antibodies, e.g.. antibodies that recognize oncological markers of cancer, including, ras. etc.
  • reagents which are specific for RCEl can be used in diagnostic and/or forensic studies according to any- desired method, e.g.. as U.S. Pat. Nos. 5.397,712; 5,434,050: 5.429,947.
  • the present invention also relates to a labelled RCEl polypeptide.
  • a labelled polypeptide can be used. e.g.. in binding assays, such as to identify substances that bind or attach to RCEl. to track the movement of RCEl in a cell, in an in vitro, in vivo, or in situ system, etc.
  • an antibody that binds to RCEl can be used to immunoprecipitate RCEl from a cell lysate to identify substances which can co-precipitate with RCEl.
  • a nucleic acid, polypeptide. antibody. RCEl ligand etc.. according to the present invention can be isolated.
  • isolated means that the material is in a form in which it is not found in its original environment, e.g., more concentrated, more purified, separated from component, etc.
  • An isolated nucleic acid includes, e.g., a nucleic acid having the sequence of RCEl separated from the chromosomal DNA found in a living animal. This nucleic acid can be part of a vector or inserted into a chromosome (by specific gene-targeting or by random integration at a position other than its normal position) and still be isolated in that it is not in a form which it is found in its natural environment.
  • a nucleic acid or polypeptide of the present invention can also be substantially purified.
  • substantially purified it is meant that nucleic acid or polypeptide is separated and is essentially free from other nucleic acids or polvpeptides. i.e.. the nucleic acid or polypeptide is the primary and active constituent.
  • the present invention also relates to a transgenic animal, e.g., a non-human-mammal, such as a mouse, comprising a RCEl nucleic acid.
  • Transgenic animals can be prepared according to known methods, including, e.g., by pronuclear injection of recombinant genes into pronuclei of 1-cell embryos, inco ⁇ orating an artificial yeast chromosome into embryonic stem cells, gene targeting methods, embryonic stem cell methodology. See. e.g., U.S. Patent Nos.
  • a nucleic acid according to the present invention can be introduced into any non-human mammal, including a mouse (Hogan et al . 1986. in Manipulating the Mouse Embryo A Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spnng Harbor. New York), pig (Hammer et al . Nature. 315 343-345. 1985). sheep (Hammer et al . Nature. 315 343-345. 1985).
  • cattle, rat. or pnmate See also, e g . Church. 1987. Trends in Biotech 5 13-19. Clark et al . 1987. Trends in Biotech 5 20-24. and DePamphi s et al , 1988.
  • nucleic acids, polvpeptides, antibodies, etc of the present invention can be prepared and used as descnbed m.
  • nucleic acids for other aspects of the nucleic acids, polvpeptides. antibodies, etc . reference is made to standard textbooks of molecular biology, protein science, and immunology See. e g . Davis et al (1986). Basic Methods in Molecular Biology, Elsevir Sciences Pubhshmg. Inc , New York. Hames et al (1985), Nucleic Acid Hybridization. IL Press, Molecular Cloning, Sambrook et al , Current
  • An assay to demonstrate RCEl can be a coupled assay linked to the prenyl-directed carboxymethvlase (yeast homolog STE14, See, e g , Methods Enzymol 1995. 250 251-66) A biotinylated, prenylated peptide substrate (e g .
  • a volume of 25 ⁇ l of membranes in 100 mM HEPES pH 7 4 is added to each well, followed by 25 ⁇ l diluted substrate (protease substrate B ⁇ ot ⁇ n-Lys-Lys-Ser-Lys-Thr-Lys-(Farnesyl)Cys-Val- He-Met is stored at -20°C in 100% DMSO but is diluted in 10% DMSO to the required working concentration immediately before use) To this is added the label I e ⁇ -SAM ( ⁇ 85C ⁇ mmol. lmCi/ml.

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